Collagen is necessary for the tensile strength and structural integrity of tissues. Cells in culture make very low levels of collagen but can be stimulated to make higher levels of collagen, in the form of procollagen, by the addition of growth factors. Additionally, cells in culture do not efficiently process procollagen to collagen. As a result, even if collagen synthesis is stimulated, almost all of the procollagen is released into the media and little or no collagen is assembled into fibrils associated with the cells.
The corneal stroma of the eye contains an extensive and transparent extracellular matrix that is interspersed with keratocytes. Transparency of this matrix requires uniformly small diameter collagen fibrils with constant inter-fibril spacing. The matrix is composed primarily of the fibrillar collagen types I and V, with lesser amounts of types III, VI and XII, and four leucine-rich type proteoglycans; three with keratin sulfate chains, lumican, keratocan, and osteoglycin/mimecan, and one with a chondroitin sulfate chain, decorin. In vitro studies have shown that collagen type V and the core proteins of the leucine-rich proteoglycans can act to regulate collagen fibril growth and these findings have been confirmed in vivo in the Col5a1 haploinsufficent mouse, in the lumican null mouse and in the keratocan null mouse.
The keratocytes in the corneal stroma are responsible for making, maintaining and repairing the corneal stroma's matrix. Keratocytes can be isolated from the stroma of rabbit and bovine corneas by collagenase digestion. Once isolated, they can be cultured in serum free media where they maintain their normal dendritic morphology as well as other in vivo characteristics (Beales et al., Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture. Invest Ophthalmol Vis Sci (1999) 40:1658-1663). The media supplement “ITS” and high levels of insulin, a component of “ITS”, have been shown to stimulate the proliferation of bovine keratocytes while maintaining their dendritic morphology and keratan sulfate proteoglycan synthesis (Musselmann K, et al., Maintenance of the keratocyte phenotype during cell proliferation stimulated by insulin. J Bio Chem (2005) 280:32634-32639). Furthermore, in the presence of ascorbic acid, a cofactor necessary for collagen triple helix stability, insulin also has been shown to increase the synthesis of collagen by 11-fold and lumican/keratocan by 2-3 fold (Musselmann et al., Stimulation of collagen synthesis by insulin and proteoglycan accumulation by ascorbate in bovine keratocytes in vitro. Invest Ophthalmol Vis Sci (2006) 47: 5260-5266). Thus, insulin may act to maintain the normal keratocyte phenotype and that proteoglycan stability may be linked to collagen stability.
Insulin has a high affinity for its own receptor, and it also has a low affinity for the IGF-I receptor. High levels of insulin would therefore activate keratocytes through both receptors. High levels of insulin would not normally be present in the corneal stroma, however IGF-II, another ligand for IGF-IR, is in the bovine corneal stroma and it causes bovine keratocytes to proliferate and maintain their normal phenotype in vitro (Musselmann et al., IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro. Exp Eye Res (2008) 86:506-511). IGF-I stimulates the proliferation of rabbit keratocytes in vitro while maintaining their dendritic morphology.
As discussed above, even if collagen synthesis is stimulated using growth factors, almost all of the procollagen is released into the media and little or no collagen is assembled into fibrils associated with the cells. Improvements in the techniques used to facilitate matrix deposition and formation are desired in the art.